Cyclooxygenase-2 mediates the development of cortical spreading depression-induced tolerance to transient focal cerebral ischemia in rats

We examined the role of cyclooxygenase-2 in the development of ischemic tolerance induced by cortical spreading depression against transient, focal brain ischemia. Cortical spreading depression was continuously induced for 2 h with topical KCl (13+/-1 depolarizations/2 h) in male Wistar rats. At 1, 2, 3, 4, and 5 days following recovery, the middle cerebral artery was transiently occluded for 120 min. Four days later, the animals were killed and infarct volume was determined. Additionally, cyclooxygenase-2 levels in the cerebral cortex and 15 deoxy-Delta(12, 14) PGJ2 levels in cerebrospinal fluid were determined at these times with Western blotting and immunoassay, respectively. Infarct volume was reduced compared with non-cortical spreading depression control animals (274.3+/-15.3 mm3) when cortical spreading depression was performed 3 and 4 days before middle cerebral artery occlusion (163.9+/-14.2 mm3, 154.9+/-14.2 mm3) but not at 1, 2 and 5 days (280.4+/-17.3 mm3, 276.3+/-16.9 mm3 and 268.5+/-17.3 mm3). Cyclooxygenase-2 levels increased most dramatically starting at 2 days, peaked at 3 days, and started to return toward baseline at 4 days after cortical spreading depression. 15 Deoxy-Delta(12, 14) PGJ2 levels increased from 134.7+/-83 pg/ml at baseline to 718+/-98 pg/ml at 3 days. Administration of N-[2-cyclohexyloxy-4-nitrophenyl] methanesulphonamide (10 mg/kg, i.v.), a selective cyclooxygenase-2 inhibitor, at 1 h prior to middle cerebral artery occlusion in cortical spreading depression preconditioned animals did not affect infarct volume (162.6+/-62.1 mm3). However, administration of N-[2-cyclohexyloxy-4-nitrophenyl] methanesulphonamide given three times prior to middle cerebral artery occlusion prevented the reduced infarct volume induced by cortical spreading depression preconditioning (272.9+/-63.2 mm3). Administration of L-nitro-arginine methyl ester (4 mg/kg, i.v.) prior to cortical spreading depression blocked increases in cyclooxygenase-2 normally seen at 3 and 4 days. We conclude that NO-mediated cyclooxygenase-2 upregulation by cortical spreading depression protects the brain against ischemic damage.     

Total microvasculature in insula and caudate nucleus following occlusion of middle cerebral artery in monkeys

Total microvasculature of insula and caudate nucleus of 36 monkeys following right middle cerebral artery occlusion was subjected to a sequential morphometric analysis with Leitz ASBA image analysis system. Total microvasculature comprising perfused functional and nonperfused reserve parts was visualized by staining alkaline phosphatase activity in the vessel wall. Number, length, and volume of visualized microvasculature increased at 0.5, 4, and 12 hours in both areas and in addition to at 24 hours in the insular cortex. Diameter and area of the microvessels were increased at 4 hours in caudate and at 0.5, 4, and 12 hours in the insular cortex followed by a decrease at 48 hours and 2 weeks in both. The intermicrovascular distance was significantly increased at 48 hours and 2 weeks in caudate. In insula it had decreased significantly at four, 12 and 24 hours and 2 weeks and increased at 48 hours following occlusion.     

Morphometric changes in perfused microvasculature in experimental focal cerebral ischemia in primates

A significant reduction in perfused microvasculature in right caudate nucleus and right insular cortex was demonstrated in 22 monkeys at 0.5, 4, 12, 24, and 48 hours and 2 weeks following transorbital occlusion of the right middle cerebral artery (RMCA). The microvasculature was visualized by an india ink perfusion method and subjected to morphometric analysis with a Leitz ASBA image analysis system. The reduction in number, length, and changes in diameter of the microvessels visualized with india ink are discussed.     

Effect of estrogen therapy on cerebral arteries during stroke in female rats

OBJECTIVE: To investigate the effect of estrogen therapy on the structural and functional properties of the middle cerebral artery during ischemia and reperfusion. DESIGN: Ovariectomized (OVX; n = 8) and ovariectomized with estrogen therapy (OVX+EST; n = 8) female Sprague-Dawley rats were exposed to 1 hour of ischemia using a model of temporary focal ischemia of the middle cerebral artery with 24 hours of reperfusion and compared to sham controls (CTL; n = 8). After occlusion and reperfusion, right middle cerebral arteries were removed from the brain and mounted on glass cannulas in a chamber that allowed for control over transmural pressure and measurement of lumen diameter. Lumen diameter was measured in response to increased transmural pressure (myogenic tone) as well as response to nitro-L-arginine, serotonin, and nifedipine. Cerebrovascular reactivity was compared to other stroke outcome measures, including infarct volume (%) and neurologic deficit. RESULTS: Serum estrogen was increased in OVX+EST rats (60.5 +/- 18.2 pg/mL) compared to OVX (0.2 +/- 0.2 pg/mL P < 0.05 vs OVX+EST) and CTL animals (1.3 +/- 1.0 pg/mL P > 0.05 vs OVX). OVX showed significantly less myogenic tone at 75 mm Hg (13.8 +/- 3.6%, P < 0.05 vs CTL) than CTL (29.8 +/- 4.7%) that was partially restored by estrogen therapy (21.2 +/- 4.5; P > 0.05). At serotonin concentrations of 10(-7) M, 3 x 10(-7) M, and 10(-6) M, the vessels from ischemic OVX rats showed significantly greater constriction (20.9 +/- 2.1%, 35.0 +/- 3.9%, and 39.4 +/- 3.4%, respectively) compared to nonischemic CTL rats (6.3 +/- 1.1%, 11.3 +/- 1.8%, and 16.8 +/- 2.5%, respectively P < 0.05). Estrogen therapy resulted in intermediate responses (18.2 +/- 5.3%, 25.2 +/- 6.6%, and 28.2 +/- 6.5%, respectively) that were not significantly different from the other groups. In addition, ischemia resulted in significantly greater dilation in response to 0.01 microM nifedipine in vessels from OVX animals (51.1 +/- 8.0%) compared to nonischemic CTL (18.0 +/- 3.8%; P < 0.05) and estrogen therapy resulted in an intermediate response (38.0 +/- 10.6; P > 0.05). Both reactivity to nitro-L-arginine and passive distensibility were not different among groups. There were no differences in percent infarct or neurologic deficit between ischemic groups. CONCLUSIONS: The influence of ischemia and reperfusion on vessel function was more dominant than that of estrogen therapy. However, estrogen therapy seemed to partially restore vessel function to similar levels as nonischemic vessels.     

Frontal cortex projections to the amygdaloid central nucleus in the rabbit

Evidence has recently been presented which demonstrates that the amygdaloid central nucleus projects directly upon cardiovascular/autonomic regulatory nuclei of the dorsal medulla and that in the rabbit this nucleus may influence cardiovascular activity during emotional states. The present study is one of a series of investigations designed to provide information on the innervation of the central nucleus in the rabbit and describes the topography and origin of frontal cortex projections to the nucleus based upon retrograde and anterograde axonal transport techniques. Injections of horseradish peroxidase or the fluorescent dyes, Bisbenzimide or Nuclear Yellow, into the central nucleus resulted in abundant numbers of retrogradely labeled neurons in three regions of the frontal cortex: the insular cortex on the lateral surface and areas 25 and 32 on the medial surface of the hemisphere. The majority of labeled neurons in the insular cortex were located in layer V of the dorsal and posterior agranular insular regions, although labeled neurons were observed in layer V of the granular insular cortex as well as in layers II and III of the posterior agranular insular cortex. Labeled neurons in areas 25 and 32 were located throughout all layers and the total number of these neurons was substantially less than that observed in the insular cortex. Autoradiographic experiments in which amino acids were injected into the insular cortex resulted in a dense pattern of transported label within the central nucleus that extended rostrally into the sublenticular substantia innominata and lateral component of the bed nucleus of the stria terminalis. Label was also observed in the cortical, lateral, basolateral and basomedial amygdaloid nuclei. In contrast to the projections from the insular cortex, amino acid injections into areas 25 and 32 resulted in only relatively light labeling within the most rostral region of the central nucleus; otherwise the nucleus was partially encapsulated and virtually devoid of label. These results suggest that the insular cortex possesses the potential to directly influence the central nucleus projection to cardiovascular/autonomic regulatory nuclei of the dorsal medulla and thus, together with the amygdaloid central nucleus, appears to be an important component of a forebrain system involved in cardiovascular/autonomic regulation.     

Responses of amygdaloid central nucleus neurons to stimulation of the insular cortex in awake rabbits

Recent evidence suggests that the amygdaloid central nucleus may contribute importantly to autonomic regulation during emotional states, possibly via both direct and indirect central nucleus efferent projections to autonomic regulatory nuclei in the lower brainstem. Additional findings suggest that the insular cortex may participate in autonomic regulatory processes, possibly by exerting an influence upon central nucleus neuronal activity via its direct projections to the central nucleus. The present experiment was conducted to determine the effects of insular cortex stimulation upon extracellularly recorded central nucleus neuronal activity in conscious, drug-free, rabbits. Satisfactory recordings were obtained from 146 central nucleus neurons. These were classified as belonging to one of six general categories on the basis of rates and patterns of ongoing discharge, responses to an auditory stimulus, and location within the nucleus. Determinations were then made as to whether each neuron could be activated antidromically from a ventrolateral mesencephalic region through which descending central nucleus projections to the lower brainstem course, and hence, whether the neuron might contribute to these projections. The activity of each neuron was then assessed during single-pulse stimulation of the region of the insular cortex demonstrated previously to project to the central nucleus. Such stimulation produced no response, an initial increase, or an initial decrease in the activity of 33%, 53% and 14% of the 146 neurons, respectively. The predominant response consisted of a single spike with a short but variable onset latency, suggesting orthodromic activation via one or a few synapses. Included among the neurons that responded to stimulation of the insular cortex in this manner were seven of 22 central nucleus neurons identified as projecting to the lower brainstem. Responses consisting of initially decreased activity most often occurred in neurons that discharged infrequently and were sensory-responsive. The results of this experiment thus provide more detailed information than was available previously regarding the pervasive influence of the insular cortex upon central nucleus neuronal activity, and provide further support for the notion that the insular cortex may participate in autonomic regulatory processes by way of its direct projections to the central nucleus.     

Resveratrol preconditioning induces cellular stress proteins and is mediated via NMDA and estrogen receptors

Resveratrol pretreatment has been shown to provide neuroprotection in models of cerebral ischemia. This phenomenon, commonly termed preconditioning, promotes ischemic tolerance and may involve mild activation of endoplasmic reticulum stress pathways in the affected tissue. Systemic injection of resveratrol (2×10⁻³, 2×10⁻⁴, 1×10⁻⁴ mg/kg) 30 min prior to a 4 h period of right middle cerebral artery occlusion significantly reduced infarct area in the insular region of rat prefrontal cortex. This affect was blocked when resveratrol treatment was combined with a non-selective estrogen receptor antagonist, or preceded by intracortical injection of an NMDA receptor antagonist. The neuroprotective effect of resveratrol was associated with reduced renal sympathetic nerve activity as well as induction of resident endoplasmic reticulum chaperone proteins, glucose-regulated proteins 78 and 94. The calcium-sensitive chaperone heat shock protein 70 and the cysteine protease m calpain did not respond to resveratrol pretreatment. However, a significant induction of heat shock protein 70 was observed in the contralateral cortex of resveratrol pretreated rats following 4 h of right middle cerebral artery occlusion. These data suggest that resveratrol preconditioning promotes ischemic tolerance in the short term, in part via effects mediated through activation of estrogen and NMDA receptors, as well as through mild activation of cellular stress proteins.     

Reduction of local cerebral blood flow to pathological levels by endothelin-1 applied to the middle cerebral artery in the rat

Endothelin-1 (1 nmol) was applied to the exposed left middle cerebral artery (MCA) in anaesthetised adult male Sprague-Dawley rats. Local cerebral blood flow (1CBF), using [14C]iodoantipyrine and quantitative autoradiography, was measured in 27 anatomically defined structures, 10 min after topical application of endothelin-1. In those areas supplied by the MCA, 1CBF was markedly reduced beyond the threshold for ischaemic damage (e.g. dorsolateral caudate nucleus reduced from 131 +/- 3 to 29 +/- 25 ml.100 g-1.min-1, sensorimotor cortex from 109 +/- 5 to 31 +/- 21 ml.100 g-1.min-1). Distant areas were not affected.     

Multiple forebrain systems converge on motor neurons innervating the thyroarytenoid muscle

The present study investigated the central connections of motor neurons innervating the thyroarytenoid laryngeal muscle that is active in swallowing, respiration and vocalization. In both intact and sympathectomized rats, the pseudorabies virus (PRV) was inoculated into the muscle. After initial infection of laryngomotor neurons in the ipsilateral loose division of the nucleus ambiguus (NA) by 3 days post-inoculation, PRV spread to the ipsilateral compact portion of the NA, the central and intermediate divisions of the nucleus tractus solitarii, the Botzinger complex, and the parvicellular reticular formation by 4 days. Infection was subsequently expanded to include the ipsilateral granular and dysgranular parietal insular cortex, the ipsilateral medial division of the central nucleus of the amygdala, the lateral, paraventricular, ventrolateral and medial preoptic nuclei of the hypothalamus (generally bilaterally), the lateral periaqueductal gray, the A7 and oral and caudal pontine nuclei. At the latest time points sampled post-inoculation (5 days), infected neurons were identified in the ipsilateral agranular insular cortex, the caudal parietal insular cortex, the anterior cingulate cortex, and the contralateral motor cortex. In the amygdala, infection had spread to the lateral central nucleus and the parvicellular portion of the basolateral nucleus. Hypothalamic infection was largely characterized by an increase in the number of infected cells in earlier infected regions though the posterior, dorsomedial, tuberomammillary and mammillary nuclei contained infected cells. Comparison with previous connectional data suggests PRV followed three interconnected systems originating in the forebrain; a bilateral system including the ventral anterior cingulate cortex, periaqueductal gray and ventral respiratory group; an ipsilateral system involving the parietal insular cortex, central nucleus of the amygdala and parvicellular reticular formation, and a minor contralateral system originating in motor cortex. Hypothalamic innervation involved several functionally specific nuclei. Overall, the data imply complex CNS control over the multi-functional thyroarytenoid muscle.     

Flow of information for emotions through temporal and orbitofrontal pathways

The posterior orbitofrontal cortex, anterior temporal sensory association areas and the amygdala have a key role in emotional processing and are robustly interconnected. By analogy with the pattern of connections in early processing sensory areas, anterior temporal sensory and polymodal association cortices send primarily feedforward projections to posterior orbitofrontal cortex and to the amygdala originating in the supragranular layers, in pathways that may provide signals about the external environment. The amygdala innervates all layers of the posterior orbitofrontal cortex, including the middle, or feedforward, target layers, in a pathway that may convey information about emotional context. The posterior orbitofrontal cortex targets dual systems in the amygdala which have opposite effects on central autonomic structures. Both pathways originate in posterior orbitofrontal cortex, but one targets heavily the inhibitory intercalated masses, whose activation can ultimately disinhibit central autonomic structures during emotional arousal. The other pathway innervates the central nucleus of the amygdala, and can lead to downstream inhibition of central autonomic structures, resulting in autonomic homeostasis. The choice of pathway may depend on emotional context, and probably involves other prefrontal areas, including lateral prefrontal areas, which have executive functions. Lateral prefrontal cortices issue feedforward projections that target layer 5 of orbitofrontal cortex, which is the chief output layer to the amygdala. These laminar-specific pathways suggest sequential and collaborative interactions in evaluating the sensory and emotional aspects of the environment for decision and action in complex behaviour.     

Afferent connections of the parabrachial nucleus in C57BL/6J mice

Although the mouse is an experimental model with an increasing importance in various fields of neuroscience, the characteristics of its central gustatory pathways have not yet been well documented. Recent electrophysiological studies using the rat and hamster have revealed that taste processing in the brainstem gustatory relays is under the strong influence of inputs from forebrain gustatory structures. In the present study, we investigated the organization of afferent projections to the mouse parabrachial nucleus (PbN), which is located at a key site between the brainstem and gustatory, viscerosensory and autonomic centers in the forebrain. We made injections of the retrograde tracer fluorogold centered around the “waist” area of the PbN, whose neurons are known to be highly responsive to taste stimuli. Retrogradely labeled neurons were found in the infralimbic, dysgranular and agranular insular cortex as well as the claustrum; the bed nucleus of the stria terminalis and the substantia innominata; the central nucleus of the amygdala; the lateral and medial preoptic areas, the paraventricular, the dorsomedial, the ventromedial, the arcuate, and the lateral hypothalamic areas; the periaqueductal gray, the substantia nigra pars compacta, and the ventral tegmental area; the supratrigeminal nucleus, rostral and caudal nucleus of the solitary tract; the parvicellular intermediate and gigantocellular reticular nucleus; the caudal and interpolar divisions of the spinal trigeminal nucleus, dorsomedial spinal trigeminal nucleus, and the area postrema. Numbers of labeled neurons in the main components of the gustatory system including the insular cortex, bed nucleus of the stria terminalis, central nucleus of the amygdala, lateral hypothalamus, and rostral nucleus of the solitary tract were quantified. These results are basically consistent with those of the previous rat and hamster studies, but some species differences were found. Functional implications of these afferent inputs are discussed with an emphasis on their role in taste.     

Fear-potentiated startle using an auditory conditioned stimulus: effect of lesions of the amygdala

The effect of lesions of the amygdala on fear-potentiated startle using an auditory conditioned stimulus (CS) was evaluated, after replicating and extending previous findings that a tone is an effective CS for fear-potentiated startle. Rats received 10 tone-shock pairings on 2 successive days. At 24-48 hr following training, they received bilateral electrolytic lesions of the central nucleus of the amygdala or sham operations, and then were tested for fear-potentiated startle 4-5 days later. Lesions of the amygdala impaired fear-potentiated startle using an auditory CS, consistent with the previous findings using a visual CS. These data indicate that the effect of lesions of the amygdala on fear-potentiated startle is not specific to one sensory modality, consistent with the hypotheses that the amygdala is involved in processing multimodal information related to conditioned fear, or is part of an output pathway for motor and autonomic expressions of conditioned fear.     

Chronic estrogen deficiency leads to molecular aberrations related to neurodegenerative changes in follitropin receptor knockout female mice

The follitropin receptor knockout (FORKO) mouse undergoes ovarian failure, thereby providing an animal model to investigate the consequences of the depletion of circulating estrogen in females. The estrogen deficiency causes marked defects in the female reproductive system, obesity, and skeletal abnormalities. In light of estrogen's known pleiotropic effects in the nervous system, our study examined the effects of genetically induced estrogen-testosterone imbalance on this system in female FORKO mice. Circulating concentrations of 17-beta-estradiol (E2) in FORKO mice are significantly decreased (FORKO -/-: 1.13+/-0.34 pg/ml; wild-type +/+: 17.6+/-3.5 pg/ml, P<0.0001, n=32-41); in contrast, testosterone levels are increased (-/-: 37.7+/-2.3 pg/ml; wild-type +/+: 3.9+/-1.7 pg/ml, P<0.005, n=25-33). The focus was on the activities of key enzymes in the central cholinergic and peripheral nervous systems, on dorsal root ganglia (DRGs) capacity for neurite outgrowth, and on the phosphorylation state of structural neurofilament (NF) proteins. Choline acetyltransferase activity was decreased in several central cholinergic structures (striatum 50+/-3%, hippocampus 24+/-2%, cortex 12+/-3%) and in DRGs (11+/-6%). Moreover, we observed aberrations in the enzymatic activities of mitogen-activated protein kinases (extracellular-regulated kinase and c-Jun N-terminal kinase) in the hippocampus, DRGs, and sciatic nerves. Hippocampal and sensory ganglia samples from FORKO mice contained hyper-phosphorylated NFs. Finally, explanted ganglia of FORKO mice displayed decreased neurite outgrowth (20-50%) under non-treated conditions and when treated with E2 (10 nM). Our results demonstrate that genetic depletion of circulating estrogen leads to biochemical and morphological changes in central and peripheral neurons, and underlie the importance of estrogen in the normal development and functioning of the nervous system. In particular, the findings suggest that an early and persisting absence of the steroid leads to neurodegenerative changes and identify several key enzymes that may contribute to the process. This model provides a system to explore the consequences of circulating estrogen deprivation and other hormonal imbalances in the nervous system.     

Connections of the dorsomedial hypothalamic nucleus from the forebrain structures in the rat

The dorsomedial hypothalamic nucleus (DMH) has been implicated as an area controlling autonomic activity. The aim of this study was to demonstrate connections of the anterior and posterior DMH to the forebrain structures, using a horseradish peroxidase (HRP) retrograde axonal transport technique in rats. The results of HRP labelling show that the anterior and posterior DMH indicate a number of differences in their connections. The posterior DMH has intense connections with the cortex (cingulate, frontal, parietal and insular), amygdala (lateral and basolateral) and hippocampus (CA1 and CA2), whereas the anterior DMH has faint connections with the cortex (cingulate, frontal and parietal) and prominent connections with the septal and bed nucleus of stria terminalis. These differences in connections of the DMH may provide sites for the specific autonomic function integrated by the DMH.     

Inputs from the olfactory bulb and olfactory cortex to the entorhinal cortex in the cat

Summary The spatial organization and laminar distribution of projections from the olfactory bulb and the anterior (PPCa) and posterior (PPCp) divisions of the prepiriform cortex to the entorhinal cortex were studied with anterograde (³H-leucine) and retrograde (WGA-HRP) tracing techniques. After ³H-leucine injections into the olfactory bulb transported labeling was seen over the lateral entorhinal area, except its most medial part, and over the rostral part of the medial entorhinal area. The labeling covers exclusively layer Ia. The lateral and medial entorhinal areas are also reached by fibers from the prepiriform cortex. The projection to the medial entorhinal area has not been described previously. Following injections of ³H-leucine into the PPCa transported labeling is present over the entire expanse of the entorhinal cortex and is located over layer Ib with the greatest density in its superficial part. Injections of ³H-leucine into the PPCp give rise to transported labeling over much of the entorhinal cortex. No labeling was found over the most medial parts of the medial subdivision (VMEA) of the lateral entorhinal area and the medial entorhinal area. Labeling occupies layer Ib, especially its middle part, and layers II and III. Both PPCa and PPCp appear to project most heavily to the dorsal (DLEA) and ventral (VLEA) subdivisions of the lateral entorhinal area. From the retrograde experiments it can be inferred that cells of layers II and III of the PPCa project predominantly to the DLEA, whereas those of the PPCp project predominantly to the VLEA. The MEA receives its heaviest projection from layer II of both PPCa and PPCp. In control experiments with ³H-leucine injections into the endopiriform nucleus it was found that this nucleus projects to the entire expanse of the entorhinal cortex. The fibers distribute to all layers with the exception of layer Ia.Abbreviations AI agranular insular cortex - AL lateral nucleus of the amygdala - BL basolateral nucleus of the amygdala - BM basomedial nucleus of the amygdala - C claustrum - CoA cortical nucleus of the amygdala - DLEA dorsal division of the lateral entorhinal cortex - END endopiriform nucleus - H hippocampus - I granular insular cortex - lot lateral olfactory tractus - MCL mitral cell layer of the olfactory bulb - MEA medial entorhinal area - OB olfactory bulb - PPCa anterior part of the prepiriform nucleus - PPCp posterior part of the prepiriform nucleus - VLEA ventral division of the lateral entorhinal cortex - VMEA ventromedial division of the lateral entorhinal cortex - 35 area 35 of the perirhinal cortex - 36 area 36 of the perirhinal cortex     

Role of corticotropin-releasing hormone in the amygdala and bed nucleus of the stria terminalis in the behavioral,pain modulatory,and endocrine consequences of opiate withdrawal

The extra-hypothalamic actions of corticotropin-releasing hormone (CRH) have been accorded an important role in coordinating responses to stressors and contributing to the consequences of drug abuse. Recent proposals suggest that CRH actions in the bed nucleus of the stria terminalis coordinate responses to tonic/unpredictable stressors whereas these actions in the central nucleus of the amygdala coordinate responses to phasic/predictable stressors. We used in situ hybridization histochemistry and site-specific microinjections of a CRH receptor antagonist to study the role of CRH in opiate withdrawal.Rats undergoing opiate withdrawal displayed clear behavioral and autonomic changes accompanied by hyperalgesia and increased plasma corticosterone. In situ hybridization of CRH mRNA revealed significant increases in the central nucleus of the amygdala but not in the bed nucleus of the stria terminalis among rats either chronically pre-treated with morphine, given an injection of naloxone, or both (precipitated withdrawal). An increase of CRH mRNA in the paraventricular nucleus of the hypothalamus was specific to rats undergoing withdrawal. Intracerebroventricular microinjection of the CRH receptor antagonist, alpha(h)CRH(9-41), reduced the severity of opiate withdrawal. Microinjections of alpha(h)CRH(9-41) into the central nucleus of the amygdala also reduced the severity of withdrawal whereas bed nucleus of the stria terminalis microinjections of alpha(h)CRH(9-41) were without effect.These experiments provide evidence for a role of amygdala, but not bed nucleus of the stria terminalis, CRH in opiate dependence. We propose a specific role for down-regulation of opiate receptor signaling in increased expression of the CRH gene in the amygdala. Moreover, we suggest that the roles accorded to CRH in the bed nucleus of the stria terminalis versus amygdala in coordinating responses to stressors may need to be reconsidered to distinguish between external and internal/interoceptive stressors.     

Ultrastructure and synaptic associations of auditory thalamo-amygdala projections in the rat

Summary Projections from the acoustic thalamus to the lateral nucleus of the amygdala (AL) have been implicated in the formation of emotional memories. In order to begin elucidating the cellular basis of emotional learning in this pathway, the ultrastructure and synaptic associations of acoustic thalamus efferents terminating in AL were studied using wheat-germ agglutinated horse-radish peroxidase (WGA-HRP) and Phaseolus vulgaris Leucoagglutinin (Pha-L) as ultrastructural anterograde axonal markers. The tracers were injected into those areas of the thalamus (medial division of the medial geniculate body and posterior intralaminar nucleus, MGM/PIN) known both to project to AL and to receive afferents from the inferior colliculus. Terminals labeled with WGA-HRP or Pha-L in AL contained mitochrondria and many small, round clear vesicles and 0–3 large, dense-core vesicles. Most labeled terminals formed asymmetric synapses on unlabeled dendrites; of these the majority were on dendritic spines. These data demonstrate that projections from the acoustic thalamus form synapses in AL and provide the first characterization of the ultrastructure and synaptic associations of sensory afferent projections to the amygdala.Abbreviations ABL basolateral nucleus of the amygdala - ABM basomedial nucleus of the amygdala - ABV ventral basolateral nucleus of the amygdala - ACE central nucleus of the amygdala - ACO cortical nucleus of the amygdala - AM medial nucleus of the amygdala - APT anterior pretectal area - AST amygdalo-striatal transition area - AL lateral nucleus of the amygdala - CI internal capsule - CG central gray - CP cerebral peduncle - CPU caudateputamen - EN endopiriform area - GP globus pallidus - I intercalated nucleus of the amygdala - OT optic tract - PIN posterior intralaminar nucleus - PIR piriform cortex - POM medial posterior thalamic complex - PP peripeduncular area - PR perirhinal cortex - SC superior colliculus - SG suprageniculate nucleus - RN red nucleus     

Relationship between metabolic dysfunctions, gene responses and delayed cell death after mild focal cerebral ischemia in mice.

The evolution of brain injury was examined in mice subjected to focal cerebral ischemia as induced by 30 min of intraluminar thread occlusion of the middle cerebral artery, followed by 3 h to 3 days of reperfusion. Metabolic dysfunctions were studied by 3H-leucine autoradiography for the measurement of cerebral protein synthesis and by regional ATP bioluminescent imaging. Metabolic changes were compared with responses of the genes c-fos, c-jun, heat-shock protein gene (hsp)72, p53-activated gene (pag)608 and caspase-3, which were investigated by in situ hybridization histochemistry and immunocytochemistry, and correlated with the degree of DNA fragmentation, as assessed by the terminal TdT-mediated dUTP-biotin nick end labeling method. Intraluminar thread occlusion led to a reproducible reduction of cerebral laser Doppler flow to 20-30% of control. Thread withdrawal was followed by a short-lasting post-ischemic hyperperfusion to approximately 120%. In non-ischemic control animals, fractional protein synthesis values of 0.81+/-0.26 and 0.94+/-0.23 were obtained. Thread occlusion resulted in a suppression of protein synthesis throughout the territory of the middle cerebral artery after 3 h of reperfusion (0.04+/-0.08 in caudate-putamen and 0.14+/-0.19 in somatosensory cortex, P<0.05). Protein synthesis partly recovered in the cortex after 24 h and 3 days (0.71+/-0.40 and 0.63+/-0.26, respectively), but remained suppressed in the caudate-putamen (0.14+/-0.22 and 0.28+/-0.28). Regional ATP levels did not show any major disturbances at the reperfusion times examined. Thread occlusion resulted in a transient increase of c-fos mRNA levels in ischemic and non-ischemic parts of the cortex and caudate-putamen at 3 h after ischemia, which suggests that spreading depressions were elicited in the tissue. At the same time, c-jun and hsp72 mRNAs were elevated only in ischemic brain areas showing inhibition of protein synthesis. C-fos and c-jun responses completely disappeared within 24 h of reperfusion. Hsp72 mRNA levels remained elevated in the cortex after 24 h, but decreased to basal values in the caudate-putamen. Twenty-four hours after reperfusion, pag608 and caspase-3 mRNA levels increased in the caudate-putamen, where protein synthesis rates were still reduced, and remained elevated even after 3 days. However, pag608 and caspase-3 mRNA levels did not increase in the cortex, where protein synthesis recovered. After 24 h and 3 days, functionally active p20 fragment of caspase-3 was detected in the caudate-putamen, closely associated with the appearance of DNA fragmented cells. Neither activated caspase-3 nor DNA fragmentation were noticed in the cortex.In summary, the suppression of protein synthesis is reversible in the ischemia-resistant cortex following 30 min of thread occlusion in mice, but persists in the vulnerable caudate-putamen. In the caudate-putamen, apoptotic programs are induced, closely in parallel with the manifestation of delayed cell death. Thus, the recovery of protein synthesis may be a major factor influencing tissue survival after transient focal ischemia.     

The pulmonary vascular response to the sustained activation of the muscle metaboreflex in man

The pulmonary circulation is influenced by the autonomic nervous system, yet whether this is physiologically important during exercise in man is not known. The aim of this study was to assess the pulmonary vascular response to sympathoexcitation induced by the maintained activation of the muscle metaboreflex in the postexercise period. Nine healthy subjects performed isometric handgrip exercise at 50% of their maximal voluntary contraction force for 2 min. Exercise was followed by circulatory occlusion so as to maintain the muscle metaboreflex activated for 2 min (postexercise circulatory occlusion; PECO). Blood pressure measurements and echocardiographically determined estimates of systolic pulmonary artery pressure (SPAP) and cardiac output were obtained at various intervals throughout the two protocols. Compared with baseline values, elevations in SPAP (by 20.06 +/- 2.08%), cardiac output (by 36.04 +/- 4.97%) and mean arterial pressure (MAP; by 25.62 +/- 3.54%) were noted during isometric exercise (means +/- s.e.m., all P < 0.05). Increases in SPAP and MAP persisted during PECO (all P < 0.05), whereas cardiac output returned to resting levels. Our findings suggest that the sympathoexcitation induced by isometric exercise affects the pulmonary circulation, possibly by inducing vasoconstriction and/or stiffening the large conduit arteries. The exaggerated activation of the sympathetic nervous system that has been evidenced in cardiopulmonary patients could therefore be implicated in their abnormal pulmonary haemodynamic responses and intolerance of exercise.     

A high soy diet reduces programmed cell death and enhances bcl-xL expression in experimental stroke

Soy phytoestrogens have been proposed as an alternative to estrogen replacement therapy and have demonstrated potential neuroprotective effects in the brain. We have shown that a high soy diet significantly reduces infarct size following permanent middle cerebral artery occlusion (MCAO). Here, we tested the hypothesis that a high soy diet would attenuate programmed cell death after stroke. Adult female Sprague-Dawley rats were ovariectomized and fed either an isoflavone-reduced diet (IFP) or a high soy diet (SP) for 2 weeks before undergoing 90 min of transient middle cerebral artery occlusion (tMCAO) followed by 22.5 h reperfusion. Infarct size, as assessed by triphenyltetrazolium chloride staining, was significantly reduced by a high soy diet (P<0.05). Apoptosis in the ischemic cortex, measured by TUNEL staining, was significantly reduced by the high soy diet. The number of active caspase-3 positive cells and caspase-mediated alpha-spectrin cleavage were also significantly decreased in the ischemic cortex of SP rats. Furthermore, nuclear translocation of apoptosis-inducing factor (AIF) was significantly reduced in the ischemic cortex of SP rats. Soy significantly increased bcl-x(L) mRNA and protein expression in the ischemic cortex compared with IFP rats. Immunohistochemistry revealed increased neuronal expression of bcl-2 and bcl-x(L) in the ischemic cortex of both IFP and SP rats following tMCAO. These results suggest that a high soy diet decreases both caspase-dependent and caspase-independent programmed cell death following tMCAO. Further, a high soy diet enhances expression of the cell survival factor bcl-x(L) following tMCAO, contributing to the neuroprotective effects of soy in the ischemic cortex.